When used as a vector, the rep & cap genes are replaced by the transgene & its associated regulatory sequences. The total length of the insert cannot greatly exceed 4.7 kb, the length of the wild type genome (Smith, 1995). Production of the recombinant vector requires that rep & cap are provided in trans, along with helper virus gene products (E1a, E1b, E2a, E4 & VA RNA from the adenovirus genome). The conventional method is to cotransfect two plasmids, one for the vector & another for rep & cap, into 293 cells infected with adenovirus (Samulski et al, 1989). This method, however, is cumbersome, low yielding (<104 particles/ml) & prone to contamination with adenovirus & wild type AAV. One of the reasons for the low yield is the inhibitory effect of the rep gene product on adenovirus replication (Vincent et al, 1997). More recent protocols remove all adenoviral structural genes & use rep resistant plasmids (Xiao et al, 1998) or conjugate a rep expression plasmid to the mature virus prior to infection (Fisher et al, 1996).
In the absence of rep, the AAV vector will only integrate at random, as a single provirus or head to tail concatamers, once the terminal repeats have been slightly degraded (Rutledge & Russell, 1997). Interest in AAV vectors has been due to their integration into the host genome allowing prolonged transgene expression. Gene transfer into vascular epithelial cells (Maeda et al, 1997), striated muscle (Fisher et al, 1997, Herzog et al, 1997) & hepatic cells (Snyder et al, 1997) has been reported, with prolonged expression when the transgene is not derived from a different species. Neutralising antibody to the AAV capsid may be detectable, but does not prevent readministration of the vector or shut down promoter activity. It is possibly due to the simplicity of the viral capsid, that the immune response is so muted. As AAV antibodies will be present in the human population this will require further investigation. There has been no attempt to target particular cell types other than by localised vector delivery.
<<< Previous Section | Next Section >>>